Heat shock/stress response proteins and prognosis in cancer

ABSTRACT

The invention relates to a method of predicting disease-free survival in cancer patients by relating the number and amount of stress response proteins in cancer tissue to the probability of tumor recurrence. Particular heat shock/stress response proteins useful in the determination of tumor recurrence are the stress response proteins, hsp70, hsp90, hsp27, and glucose regulated protein grp94. Specific levels of the stress response proteins relative to an internal standard are identified, above which the probability of tumor recurrence is highly significant. Kit methods are disclosed which could enable determination of the stress proteins by an antibody assay.

The United States Government may have certain rights in the presentinvention pursuant to the terms of Grant No. CA 11378 awarded by theNational Cancer Institute.

This is a continuation-in-part of U.S. patent application Ser. No.07/509,377 filed Apr. 12, 1990, now U.S. Pat. No. 5,188,964.

The invention relates to a method of predicting disease-free survival incancer patients based on overproduction levels of one or more stressresponse proteins from primary tumors. The method is particularly usefulin predicting tumor recurrence in node-negative breast cancers.

Stress response proteins, srp's, have been recognized for several years,although in earlier terminology they were commonly called heat shockproteins, hsp's, because they were originally discovered as families ofrelated proteins rapidly overproduced in divergent species in responseto temperature stress. Subsequently these proteins were found to beinduced in response to a variety of environmental stresses, includingstimuli such as heat, heavy metals, toxins, drugs, hypoxia, and alcohol.

The precise function of stress response proteins is still largely amatter of speculation. It is widely assumed that these proteins protectcells from the effects of stress, but little is known about themechanisms of induction and even less is understood about therelationships between number and amount of protein induced and theunderlying physiological phenomenon.

There have been speculations that pretumorous or tumor cells mightexpress increased amounts of stress response proteins, leading someworkers to search for a relation between levels of these proteins andtumor manifestation. But although readily induced, the higher levels ofheat stress proteins did not appear to relate to increased probabilityof tumor recurrence; in fact, some studies indicated that metastatictumor burden generally decreased following induction of stress proteins(S. P. Tomasovic and D. R. Welch, Hyperthermia 2, 253 (1986)).Subsequent work by McGuire and colleagues, however, demonstrated that anestrogen-induced protein found in MCF-7 human breast cancer cells wasidentical to one of the earlier discovered heat shock proteins, hsp27,and that hsp27 might be associated with node-negative breast cancerpatients at high risk of recurrence. Nevertheless, correlation factorswere relatively weak and not sufficient to suggest a clinically usefulmethod of prognostication.

The phenomenon of heat shock response was first observed nearly threedecades ago by Ritossa (F. Ritossa, Experientia 18, 571 (1962)) whofound that an increase in temperature from 20° to 37° C. as well asexposure to certain chemicals such as dinitrophenol or sodiumsalicylate, leads to a remarkable change in the puffing pattern ofpolytene chromosomes in salivary glands of fruit flies (Drosophilabusckii). Nearly 12 years after this observation, Tissieres et al. (A.Tissieres, H. K. Mitchell, U. M. Tracy, J. Mol. Biol. 84, 389 (1974))reported the induction of a set of proteins called heat shock proteins(hsp's), as a consequence of heat shock. Today, practically all types oforganisms are known to respond to an increase in temperature in abasically similar fashion by massive synthesis and accumulation of agroup of hsp's with almost no tissue or cell type specificity. Majorhsp's are now known to be very highly conserved through evolution,strongly suggesting their vital role in survival of the organisms.Nearly all species induce the synthesis of proteins in the size rangesof 80 to 90 kDa, 68 to 74 kDa, and 18 to 30 kDa. In the past few years,the genes encoding the hsp's have been isolated and through sequenceanalysis have been placed into three "universal" families. These areknown by their molecular weights: hsp90, hsp70, and hsp27 (the exactmolecular weight differs slightly from organism to organism). These hspgenes contain a conserved sequence of 14 base pairs in the 5' noncodingregion, the Pelham box, which serves as the promoter for hsp mRNAtranscription. Recently, a relationship between the sequences of hsp'sand another family of stress proteins, the glucose-regulated proteins(grp's), has been reported. These proteins are oversynthesized inresponse to glucose starvation. Two major grp's have been identified asgrp94 and grp78.

Although the precise function of stress response proteins (srp's) is notknown, they are thought to be intimately involved in enhancing thecell's ability to recover from stress (e.g. conferring thermotolerance).Yet the exact biochemical mechanism of this protection of cells againstphysical and chemical environmental insults remains a mystery. There areseveral excellent and comprehensive reviews on this subject includingthe organization and regulation of expression of hsp genes (S. Linquist,E. A. Craig, Ann. Rev. Genet. 22, 631 (1988); M. J. Schlesinger, J. CellBiol. 103, 321 (1986); H. R. B. Pelham, Cell 46, 959 (1988); E. A.Craig, CRC Crit. Rev. Biochem. 18, 239 (1985)).

In 1980, a cytoplasmic, estrogen-induced protein of 24,000-28,000Daltons molecular weight (termed "24K") in MCF-7 human breast cancercells (D. P. Edwards, D. J. Adams, N. Savage, W. L. McGuire, Biochem.Res. Commun. 93, 804 (1980)) was reported. Its relative abundance inestrogen-stimulated MCF-7 cells enabled researchers to rapidly develop ahighly specific monoclonal antibody against it (D. J. Adams, H. Hajj, D.P. Edwards, R. J. Bjercke, W. L. McGuire, Cancer Res. 43, 4297 (1983)).Nucleotide and deduced amino acid sequence of "24K" (S. A. W. Fuqua, M.B. Salingaros, W. L. McGuire, ibid. 4.9, 4126 (1989)) revealed itsidentity to the low molecular weight human heat shock protein hsp27,earlier reported in HeLa cells (E. Hickey et al., Nucleic Acid Res. 14,4127 (1986)). Somatic cell hybridization showed that it is a multigenefamily, located on three different chromosomes namely 3,9 and X (S.McGuire, S. A. W. Fuqua, S. L. Naylor, D. A. Helen-Davis, W. L. McGuire,Somatic Cell Genet. 15, 167 (1989)). It is dually induced by heat shockas well as by estrogen in MCF-7 cells. A study of its possiblesignificance for predicting clinical outcome showed that it was a factorfor defining node-negative breast cancer patients at high risk ofrecurrence (G. C. Chamness, A. Ruiz, L. Fulcher, G. M. Clark, W. L.McGuire, Breast Cancer Res. Treat. 12, 130 (1988) (Abstract #94); G. C.Chamness et al., Proc. Am. Assoc. Cancer Res. 30, 252 (1989)) (Abstract#1002).

The heat shock protein hsp90 is known to interact with severalprotein-tyrosine kinases between the time of their synthesis and theirultimate association with the plasma membrane. The transforming proteinof Rous Sarcoma Virus, pp60^(src), was the first tyrosine kinase withwhich hsp90 was shown to have a specific association (J. S. Brugge, E.Erikson, R. L. Erikson, Cell 25, 363 (1981); H. Oppermann, W. Levinson,J. M. Bishop, Proc. Natl. Acad. Sci. 78, 1067 (1981)). Othertransforming proteins with tyrosine kinase activity, yes, fps, fes, andfgr, also form stable complexes with a 90 kDa protein. In some casesthis 90 kDa protein has been identified as hsp90 (B. Adkins, T. Hunter,B. M. Sefton, J. Virol. 43, 448 (1982); L. A. Lipsich, J. R. Cutt, J. S.Brugge, Mol. Cell Biol. 2, 875 (1982); and A. Ziemiecki, Virology 151,265 (1986)). It has been proposed that hsp90 transports and modulatesthese kinases by forming soluble, inactive complexes. Hsp90 has alsobeen found associated with other cellular kinases, e.g. heme-controlledelF2-alpha kinase and casein kinase II (D. W. Rose, R. E. H. Wettenhall,W. Kudlicki, G. Kramer, B. Hardesty, Biochemistry 26, 6583 (1987)).

All steroid hormone receptors, including the estrogen, progesterone, andglucocorticoid receptors, can be isolated in the inactivated state(i,e,., in the absence of steroid hormones) as approximately 300 kDacomplexes, which in addition to the specific hormone-binding proteins,contain 90 kDa proteins that have now been identified as hsp90 (M. G.Catelli, C. Radanyi, J. M. Renoir, N. Binart, E. E. Baulieu, J. CellBiochem. Suppl. 12D, 286 (1988); J. J. Dougherty, R. K. Puri, D. O.Toft, J. Biol. Chem. 259, 8004 (1984); I. Joab, et al., Nature 308, 850(1984); G. Redeuilh, B. Moncharmont, C. Secco, E.-E. Baulieu, J. Biol.Chem. 262, 6969 (1987); J.-M. Renoir, T. Buchou, E.-E. Baulieu,Biochemistry 25, 6405 (1986); and E. R. Sanchez, P. R. Housley, W. B.Pratt, J. Steroid Biochem. 24, 9 (1986)). Dissociation of hsp90 from thecomplex leads to the activation of the receptor for DNA binding (I.Joab, et al., Nature 308, 850 (1984); J.-M. Renoir, T. Buchou, E.-E.Baulieu, Biochemistry 25, 6405 (1986); and E. R. Sanchez, et al., J.Biol. Chem. 262, 6986 (1987)). In the absence of hsp90, thehormone-binding receptor will bind to the DNA whether hormone is presentor not (E. R. Sanchez, et al., J. Biol. Chem. 262, 6986 (1987)). Hsp90itself binds neither DNA nor hormone. Apparently, binding of hsp90 tothe receptor prevents the receptor from binding to DNA until hormonedisrupts association of hsp90 to the receptor.

In broad outline, hsp90 appears to play a role in steroid receptorcomplexes similar to that in tyrosine kinase complexes, keeping thereceptor inactive until the proper signal for activation is received.

Recently, hsp90 has also been reported to associate with actin inlymphocyte extracts, in a manner dependent on calcium and regulated bycalmodulin (S. Koyasu, et al., Proc. Natl. Acad. Sci. U.S.A. 83, 8054(1986); and E. Nishida, S. Koyasu, H. Sakai, I. Yahara, J. Biol. Chem.261, 16033 (1986)). It has been postulated that the actin associationprovides a mechanism for transport of hsp90. In this regard, andconsidering the tendency of hsp90 to move into the nucleus with heatshock, it is notable that actin filaments rearrange during heat shockand may even be found in substantial quantities in the nuclei ofheat-shocked cells (W. J. Welch, J. P. Suhan, J. Cell Biol. 101, 1198(1985)). Hsp90 also appears to be associated with tubulin both in vitroand in vivo (E. H. Bresnick, T. Redmond, E. R. Sanchez, W. B. Pratt, M.J. Welsh, J. Cell Biochem. Suppl. 12D, 283 (1988)). Given the highconcentrations of actin, tubulin, and hsp90 in the cell, theseassociations may be biologically significant.

A tumor-specific transplantation antigen, Meth A, has also recently beenidentified as hsp90 (S. J. Ullrich, E. A. Robinson, L. W. Law, M.Willingham, E. Appella, Proc. Natl. Acad. Sci. U.S.A. 83, 3121 (1986)).

In humans, the heat shock protein hsp70 represents a multigene family,located on chromosomes 6, 14, 21, and at least one other chromosome (A.M. Goate, et al., Mum. Genet. 75, 123 (1987); and G. S. Harrison, etal., Somatic Cell Mol. Genet. 13, 119 (1987)). Their protein productsare present in different cellular compartments and are often associatedwith other proteins. All bind ATP with high affinity (T. G. Chappell, etal., Cell 45, 3 (1986); W. J. Welch, J. R. Feramisco, Mol. Cell. Biol.5, 1229 (1985); and M. Zylicz, J. H. LeBowitz, R. McMacken, C. P.Georgopoulos, Proc. Natl. Acad. Sci. U.S.A. 80, 6431 (1983)) and areimplicated in a number of cellular processes. The major hsp70 is a cellcycle regulated protein (K. L. Milarski, R. Morimoto, Proc. Natl. Acad.Sci. U.S.A. 83, 9517 (1986)), is serum stimulated (B. J. Wu, R. I.Morimoto, Proc. Natl. Acad. Sci. U.S.A. 82, 6070 (1985)), and is inducedby adenovirus E1A protein (J. R. Nevins, Cell 29, 913 (1982)).

It seems that the cell exploits a general property of the hsp70 family,namely the ability to disrupt protein-protein interactions, to performspecific tasks. Most of the "reactions" involving proteins of the hsp70family require ATP. Probably the disruption of the protein-proteininteractions uses the energy released on ATP hydrolysis.

One function of hsp70 may be the repair of damaged cells. Very shortlyfollowing heat shock, hsp70 translocates from cytoplasm to nucleuschanging from a "soluble" cytosolic to an "insoluble" nuclear-matrixform. In the nucleus, it subsequently concentrates in nucleoli where itapparently binds to partially assembled ribosomes (W. J. Welch, J. P.Suhan, J. Cell Biol. 103, 2035 (1986)). Nucleoli are very sensitive tothermal damage, but transfection of cells with a plasmid thatoverproduces hsp70 accelerates their recovery from heat shock (H. R. B.Pelham, EMBO J 4, 3095 (1984)), indicating that hsp70 binds to denaturedor abnormal proteins after heat shock to prevent their aggregation andthus to prevent cellular damage. Hsp70 is rapidly and completelyreleased from "insoluble" nuclear matrix on addition of ATP (M. J.Lewis, H. R. B. Pelham, EMBO 4, 3137 (1985)). This observation led Lewisand Pelham (M. J. Lewis, H. R. B. Pelham, EMBO J 4, 3137 (1985)) topropose that hsp70 binds to denatured, aggregated proteins andsolubilizes them. Energy from ATP hydrolysis subsequently causes hsp70to release, thereby allowing the proteins to refold.

Clathrin-uncoating ATPase has been identified as a member of the hsp70gene family. In the presence of ATP, an hsp70-like protein binds to theclathrin cages and is induced to hydrolyze ATP, resulting in thedisruption of clathrin-clathrin interactions and finally in disassemblyof the cage into clathrin trimers (J. E. Rothman, S. L. Schmid, Cell 46,5 (1986); T. G. Chappell, et al., Cell 45, 3 (1986); and E. Ungewickell,EMBO J 4, a385 (1985)).

In E. coli, hsp70 is the product of the dnak gene, which encodes aprotein that is 50% identical in amino acid sequence to hsp70 ofeukaryotes (J. C. A. Bardwell, E. A. Craig, Proc. Natl. Acad. Sci.U.S.A. 81, 848 (1984)). Dnak protein interacts with lambda phage O and Pproteins during phage replication (J. H. LeBowitz, C. Zylicz, C.Georgopoulos, R. McMacken, Proc. Natl. Acad. Sci. U.S.A. 82, 3988(1985); and Dodson et al., Proc. Natl. Acad. Sci. U.S.A. 82, 4678(1985)), again implicating hsp70 in the disruption of a tightprotein-protein interaction. Like clathrin uncoating, this is an exampleof a specific function that exploits the general properties of thehsp70-like proteins.

In cells that overproduce the transformation-associated protein p53,stable complexes form between p53 and hsp70-related proteins (O.Pinhasi-Kimhi, D. Michalovitz, A. Ben-Zeev, M. Oren, Nature 320, 182(1986)). Mutations in the gene encoding p53 that inactivate its tumorsuppressing potential also result in the synthesis of mutant proteinswhich show preferential association with hsp70-like proteins and have anincreased half-life (C. A. Findley, et al., Mol. Cell. Biol. 8, 531(1988)). It is hypothesized that this interaction leads to a higherstability of p53, and the complex can be dissociated in vitro with ATP.Interestingly, p53 synthesized in E. coli is found in association withdnak protein (C. F. Clarke, et al., Mol. Cell. Biol. 8, 1206 (1988)).

Several eukaryotic cell DNA viruses, i.e., adenovirus (J. R. Nevins,Cell 29, 913 (1982)), herpes virus (E. L. Notarianni, C. M. Preston,Virology 123, 113 (1982)), and Simian Virus 40 and polyoma viruses (E.W. Khanjian, H. Turler, Mol. Cell. Biol. 3, 1 (1983)) activate synthesisof hsp70 early in infection. Newcastle Disease Virus, an RNA virus,induces hsp70 and hsp90 in infected chicken cells (P. C. Collins, L. E.Hightower, J. Virol. 44, 703 (1982)). Hsp70 itself is reported to have aprotease activity (H. K. Mitchell, N. S. Petersen, C. H. Buzin, Proc.Natl. Acad. Sci. U.S.A. 82 4969 (1985)).

Two proteins related to hsp70 and hsp90 and regulated by glucosestarvation have been identified as grp78 and grp94, respectively (A. S.Lee, J. Bell, J. Ting, J. Biol. Chem. 259, 4616 (1984); and R. P. C.Shiu, J. Pouyssegur, I. Pastan, Proc. Natl. Acad. Sci. U.S.A. 74, 3840(1977)). Grp's are not normally heat-inducible, but are overproducedunder a variety of other physiological stresses such as anoxia,paramyxovirus infection, and treatment of cells with glycosylationinhibitors (S. C. Chang, et al., Proc. Natl. Acad. Sci. U.S.A. 84, 680(1987)) or the calcium ionophore A23187 (Lin et al., Mol. Cell Biol. 6,1235 (1986)). These proteins are abundant in secretory cells and arefound associated with endoplasmic reticulum, and may possibly carry outthe same functions as hsp's.

Grp78 is about 60% homologous to hsp70 and is identical to BiP (S.Munro, H. Pelham, Cell 46, 291 (1986)), a protein known to bind to theimmunoglobulin heavy chains in pre-B cells that do not make light chains(Bole et al., J. Cell Biol., 102, 1558 (1986)). This finding suggeststhat grp78 prevents the formation of heavy chain aggregate and thushelps the process of immunoglobulin assembly. Grp78 binds to theaberrant proteins to keep them soluble in the same way as hsp70 acts onheat-denatured nuclear proteins. For example, it associates with mutantsof hemagglutinin of influenza virus that fail to assemble into a maturetrimeric glycoprotein (M. J. Gething, K. McCammon, J. Sambrook, Cell 46,939 (1986)). Mammalian cell lines with decreased amounts of grp78 showincreased secretion of mutant proteins (A. Dorner, M. Krane, R. Kaufman,J. Cell. Biochem. suppl. 12D, 276 (1988)).

Grp94 has been partially sequenced, showing that the protein is morethan 50% homologous to yeast hsp90 and Drosophila hsp83 (P. K. Sorger,H. R. B. Pelham, J. Mol. Biol. 194, 341 (1987)). It is glycosylated,soluble in the absence of detergents, and is probably a luminal protein.The role of grp94 is even less understood than that of grp78.

There is also little information on the function of the low molecularweight hsp's. A stretch of 75 amino acids which are conserved among foursmall Drosophila hsp's is found to be 50% homologous to the B chain ofmammalian lens alpha crystallins (H. Bloemendal, T. Berns, A. Zweers, H.Hoenders, E. L. Benedetti, Eur. J. Biochem. 24, 401 (1972); and T. D.Ingolia, E. A. Craig, Proc. Natl. Acad. Sci. U.S.A. 79, 2360 (1982)),suggesting that these hsp's may serve some kind of structural role. TheDrosophila hsp's form large insoluble aggregates in a perinuclear regionof the cell after prolonged heat shock (N. C. Collier, M. J.Schlesinger, J. Cell Biol., 103, 1495 (1986); and L. Nover, K.-D.Scharf, D. Neumann, Mol. Cell. Biol. 3, 1648 (1983)), but theseaggregates dissociate during cell recovery.

There is also an association of srp's with acquired drug resistance.Exposure of renal adenocarcinoma cells to heat shock or chemicalstresses has shown that the major MDR1 gene promoter has heat shockelements and its expression (both mRNA and protein) is increased bythese stresses, with a concomitant development of resistance tovinblastine (K.-V. Chin, S. Tanaka, G. Darlington, I. Pastan, M. M.Gottesman, J. Biol. Chem. 265, 221 (1990)). MDR1 RNA levels, however,did not change following stresses that normally induce grp's. Similarly,in Chinese hamster ovary cells, Shen et al. (J. Shen, et al., Proc.Natl. Acad. Sci. U.S.A. 84, 3278 (1987)) found that the induction ofgrp's did not change the level of MDR1-encoded P-glycoprotein. But thesecells nevertheless acquired resistance to doxorubicin through an unknownmechanism. These observations suggest that some srp's may also beinvolved either indirectly or directly in conferring drug resistance tocells. In renal cells, MDR1-encoded P-glycoprotein may additionallyprotect cells from the effects of heat shock and chemical stresses.

More recently, Huot et al. (personal communication) found thattransfection of Chinese hamster ovary cells with the hsp27 gene resultsin development of multidrug resistance. These studies indicate the roleof a specific hsp in the phenomenon of multidrug resistance.

Prognosis in clinical cancer is an area of great concern and interest.It is important to know the aggressiveness of the malignant cells andthe likelihood of tumor recurrence in order to plan the most effectivetherapy. Breast cancer, for example, is managed by several alternativestrategies. In some cases local-regional and systemic radiation therapyis utilized while in other cases mastectomy and chemotherapy ormastectomy and radiation therapy are employed. Current treatmentdecisions for individual breast cancer patients are frequently based on(1) the number of axillary lymph nodes involved with disease, (2)estrogen receptor and progesterone receptor status, (3) the size of theprimary tumor, and (4) stage of disease at diagnosis (G. M. Clark etal., N. Engl. J. Med. 309, 1343 (1983)). It has also been reported thatDNA aneuploidy and proliferative rate (percent S-phase) can help inpredicting the course of disease (L. G. Dressler et al., Cancer 61, 420(1988); and G. M. Clark et al., N. Engl. J. Med. 320, 627 (1989)).However, even with these additional factors, we are still unable toaccurately predict the course of disease for all breast cancer patients.There is clearly a need to identify new markers, in order to separatepatients with good prognosis who will need no further therapy from thosemore likely to recur who might benefit from more intensive treatments.

This is particularly true in the case of breast cancer which has notprogressed to the axillary lymph nodes. There is now evidence inprospective randomized clinical trials that adjuvant endocrine therapyand adjuvant chemotherapy beginning immediately after surgical removalof the primary breast tumor can be of benefit in some of thesenode-negative patients. This has led to official and unofficialrecommendations that most if not all node-negative breast cancerpatients should be considered for some form of adjuvant therapy. Butsince the majority (˜70%) of these patients enjoy long-term survivalfollowing surgery and/or radiotherapy without further treatment, it maybe inappropriate to recommend adjuvant therapy for all of thesepatients. If there were sufficiently good methods to distinguish thosenode-negative patients who are "cured" from those destined to recur,only the latter should be treated. Thus, there is a great need for ageneral method of predicting tumor recurrence in these patients and incancer patients in general once the primary tumor is detected.

The present invention seeks to address one or more of the foregoingproblems in predicting tumor recurrence when the mere presence of anunusual protein is insufficient to reliably predict a present orpotential disease state. It has been found that the number and"overproduction" levels of stress response proteins in primary tumortissue show an unexpected and surprisingly high correlation with tumorrecurrence. Consequently, the present work represents a significantadvancement in cancer management because early identification ofpatients at risk for tumor recurrence will permit aggressive earlytreatment with significantly enhanced potential for survival.

It should be recognized that stress response proteins (srp's) are aclass of proteins of which the previously recognized heat shock proteins(hsp's) are a subclass, and the glucose response (or glucose regulated)proteins (grp's) are another subclass. Since the hsp's respond to manystresses besides heat, there is a trend toward using the genericdesignation srp's for these proteins. In the present invention, srp'swill refer to the class, while specific proteins will be given theiroriginal designations (e.g. hsp27).

The stress response proteins used in one embodiment of the invention areheat shock proteins 27, 70 and 90 and glucose response protein 94. Theseare known in the art as members of families of proteins having molecularweights in humans in the range of 27,70,90 and 94 kDa respectively. Itis intended that stress response proteins refer to any stress responseprotein produced in the tumor cell for which it would be possible, usingthe method of the present invention, to determine "overproduction"levels. As yet undiscovered or untested stress response proteins wouldalso be expected to be correlated with tumor recurrence since they areproduced in response to the same stresses that result in hsp27, 70, 90and/or grp94 overproduction. Up to four stress response proteins havebeen associated with tumor recurrence, but since correlation increaseswith the number of stress response proteins, it is expected thatdetection of additional stress response proteins will further improvecorrelation.

In one embodiment of the invention, the presence of two or more of theseproteins can be correlated with tumor recurrence. However, generally themere presence of the proteins is relatively weakly associated with tumorrecurrence only when several of the proteins are detected, for example,when four srps are present. Stronger correlations have been found whenthe levels of the proteins exceed "overproduction" levels. Such"overproduction" is not typically calculated in terms of absoluteprotein levels, but is determined using relative measurements. Theserelative measurements are illustrated for quantitation purposes with an"internal standard"; however, it will be appreciated that otherstandards or methods of determination may be used, such as comparisonexternal standards, hsp mRNA measurements, or absolute values.

One measure of relative value of "overproduction" is to determinerelationships of tumor sample stress response protein levels to basallevel of stress response proteins produced by a cultured cell line. Mostcultured cells produce srps, probably because of particular stressesimposed by culture conditions. Normal cells, cancer cells, orgenetically altered cells could be used. Although human cells arepreferred, bacterial, yeast, other mammalian cells or the like alsoproduce similar stress-induced proteins which in fact often showsignificant homology with human stress-induced counterparts (Watowich,S. S. and Morimoto, R. I., Mol. Cell. Biol 8, 393-404 (1988)). Cellsparticularly sensitive to stress are preferred, while breast cancercells have been particularly useful. A most preferred cell line is astandard breast tumor cell line on deposit with the American TypeCulture Collection (ATCC, MCF-7).

A basis for one aspect of the present invention is the inventors'finding that "overproduction" levels of stress response proteins areassociated with tumor recurrence. When a stress response protein exceedsa determined basal level, it has been found to become a significantfactor in tumor recurrence. It is important to recognize that basallevels are not the levels of srps found in cultured cell lines used toprovide internal standards; rather, these are amounts of srps which maybe found in tumor cells that do not cause tumor recurrence. It is, forexample, possible that an "overproduced" tumor sample stress responseprotein will have a relatively lower level than the corresponding srp inthe internal standard. When tumor cell determined basal levels areexceeded, the stress response is "overproduced" and indicates increasedrisk of tumor recurrence. On the other hand, the mere presence, withoutoverproduction, of one or more stress response proteins is rarelycorrelated with higher risk of tumor recurrence.

Overproduction is related to a level of srp's above a determined low orbasal level and is different for each srp. Thus, in this invention alevel of each protein is identified as a "cutoff" value, above whichthere is a significant correlation between the presence of the srp andtumor recurrence. Some "cutoff" values are not sharp in that clinicalcorrelations are still significant over a range of values on either sideof the cutoff; however, it is possible to select an optimal cutoff valuefor each stress response protein. The cutoff value used for a givenapplication is termed the "cutpoint".

Generally, overproduction of several stress response proteins provideshigher correlations with tumor recurrence than overproduction of one ortwo stress response proteins. In one embodiment of the invention, anyone of grp94, hsp70 or hsp90 may be used to correlate tumor recurrencewith overproduction levels. In a preferred embodiment, any two or threeof hsps selected from hsp70, hsp90, hsp27 or grp94 may be used. In amost preferred embodiment, overproduction levels of hsp27, hsp70, hsp90and grp94 are measured.

Measurement of levels of grp94 and three hsp's (hsp90, hsp70, and hsp27)in a large cohort of well characterized human primary breast tumors withextensive clinical followup, has demonstrated that overproduction ofthese proteins occurs relatively frequently in breast cancer, and,surprisingly, with considerable correlation among the four srp's.Furthermore, higher levels of all (grp94 marginally) are associated withtumor recurrence behavior in breast cancer patients having no tumorextensions to the axillary lymph nodes at primary treatment.Simultaneous occurrence of more than one srp is a more powerfulpredictor of poor disease-free survival; the higher the number ofoverproduced srp's, the greater is the risk of tumor recurrence.

A relative measure of "overproduction" is used in the practice of thisinvention. The units defining "overproduction" are relative to anarbitrarily assigned cancer cell line standard. In a preferredembodiment, MCF-7 human breast cancer cells (ATCC-HTB22 MCF-7) are grownunder defined conditions. Aliquots of homogenized cells are analyzedalong with each set of samples of test tissue by standard procedures.Each μg protein aliquot of MCF-7 cells is arbitrarily assigned a "oneunit" value so that the level of any given stress response protein inthe test sample is measured in units against this standard MCF-7 cellunit. Absolute values of the arbitrary "units" can be readily determinedby one skilled in the art. It should be appreciated that the"overproduction" values selected are illustrative and that correlationsmay still exist between levels of stress response proteins fallingsomewhat below the given cutoff values and tumor recurrence. Selectionof the cutoff values is determined by statistical considerations anddoes not imply an absolute value.

Correlations were determined by well known multivariate analysistechniques. Different statistical treatment could be used to defineother sets of values for the stress response proteins which couldimprove correlations. For example, if improved correlations were found,overproduction values would differ somewhat from the cutoff valuesdefined in the present invention. In the present invention, ranges ofvalues were determined for each stress response protein. In a preferredembodiment, optimal cutoff levels of the stress response proteins hsp27,70, and 90 and grp94 are 126, 217, 32, and 45 units per 100 μg tumorprotein. Above these levels, the proteins are considered overproducedand hence indicative of significant risk of tumor recurrence. It isunderstood that improvements in optimal cutoff values could bedetermined, depending on the sophistication of statistical methods usedand on the number and source of samples used to determine basal valuesfor the different hsps.

The stress response proteins useful in the practice of this inventionare typically produced in response to cell stress. The four used in thepresent invention are the best known, but others have been discoveredand are contemplated as being included in the invention. It is likely,for example, that grp78, another glucose response protein, will exhibitcorrelation with tumor recurrence when overproduced in cancer tissue.The details of measurement and correlation of grp78 would be analogousto those used in the present invention to determine overproduction ofgrp94 and hsp's 27, 70 and 90.

In preferred practice of the invention, hsps are extracted from tumorcell tissue. However, it should also be possible to measure levels ofthese proteins in the serum. Tumors are known to readily shed cells and,after release into the bloodstream might be expected to burst due tocell fragility. Thus detection of any hsps present could be used todevelop overproduction correlations in a manner analogous to thatdemonstrated with the tissue samples. Very small quantities of hspsmight be measured, for example, using antibodies directed to theparticular hsps.

Immunologically based diagnostic kits for determining the number andlevels of stress response proteins in tissue samples are contemplated.Such kits would include the appropriate anti-srp antibody together withan immunoreaction detection reagent. As used herein, an immunoreactiondetection reagent is a reagent capable of detecting or indicating aspecific immunoreaction between the antibody and the hsp antigen.Examples of such reagents include enzyme, fluorescent or radiolabeledantigens or antibodies. Many such reagents and their use are well knownto those of skill in the art. Some of the methods of detection utilizingimmunoreactions include Western blot, immunohistochemical procedures andELIZA assays. These methods are also well known to those skilled in theart.

An example of kit components would include a stress response proteinstandard with several stress response proteins, antibodies (preferablymonoclonal) to each stress response protein, a negative control and apositive control. An example of a control is a breast tumor extract, butother standards could be used, including synthetic standards preparedfrom synthesis of all or part of the srps of interest. Tumor cellextracts are preferable, however, as the presence of other cellcomponents would more closely mimic the sample. For convenience, thesecomponents may be supplied in lyophilized form.

Several variations of kits based on antibody binding are envisioned. Forexample, use of a second antibody specifically directed against thefirst antibody in a sandwich type detection system can be used inwell-known variations of the ELISA technique. Detection of secondantibodies tagged with various labels, including radioisotopes,chromophores, enzymes, and the like could also be used. Anothervariation is the binding of an antibody, for example a monoclonalantibody, by a second biotinylated antibody followed by reaction with anavidin biotinylated horseradish peroxidase complex. If the firstantibody is attached to a substrate such as an agarose bead or amicrotiter plate well, the color developed can be quantitated relativeto a standard supplied in the kit.

Although the applications of the invention which are described here arebased on antibodies specific for each srp, it will be understood bythose in the art that the level of each srp is related to the level ofthe messenger RNA (mRNA) which encodes it. Therefore, when the aminoacid sequence of the srp is known, methods can easily be envisioned bywhich srp overproduction in tumors would be determined by measuringlevels of the corresponding mRNA's. Rather than antibodies,complementary DNA's for each srp mRNA would be the specific recognitionelements, and the existing techniques known as Northern blots, slotblots, in situ hybridizations, and polymerase chain reactions (PCR)would be applied. Messenger RNA levels have been used to determineproduction of corresponding proteins (G. Bevilacqua, M. E. Sobel, L. A.Liotta and T. S. Steeg, Cancer Res. 49, 5185-5190 (1989)). Amino acidsequences, and consequent cDNA preparation, for several of the srps areknown, including hsp70 (Watowich, S. S. and Morimoto, R. I., Mol. Cell.Biol., 8, 393-405 (1988)), hsp90 (Hickey, L., Brandon, S. E., Smale, G.,Lloyd, D. and Weber, L. A., Mol Cell 9, 2615-2625 (1989)) and hsp27(Hickey, et al., Nucleic Acids Res. 14, 4127-4145 (1986).

FIG. 1 shows a Western Blot analysis of ten human breast tumor extractsusing monoclonal antibodies to hsp27, hsp70, hsp90, and grp94. Aninternal reference standard of an MCF-7 human breast cancer cell proteinextract is shown as the standard against which relative units werecalculated.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H are a series of graphs showingdistributions of stress response proteins in node-negative andnode-positive breast cancer patients, and the statistical significanceof the full range of possible cutoff values for each srp in predictingrecurrence.

FIGS. 3A, 3B, 3C and 3D show the association of stress response proteinswith tumor recurrence for node-negative breast cancer patients using aKaplan-Meier analysis of time to recurrence.

FIG. 4 illustrates Kaplan-Meier recurrence curves for node-negativebreast cancer patients based on higher levels of one or more of thestress response proteins.

The present invention allows prediction of survival or tumor recurrencein cancer patients based on a determination of one or more stressresponse proteins (srps) in a tumor sample. The mere presence of thesesrps in tumor tissue has little prognostic value; however, surprisingly,when one or more srp exceeds a determined basal level in the tumortissue, good correlations with tumor recurrence have been found.Further, in another unexpected finding, the greater the number of"overproduced" srps, the better the correlation with long-term (greaterthan five years) survival.

The method is described here in detail for determination of"overproduction" in breast tumor samples using a relative measure ofsrps. The relative measure is based on comparison with srp production incultured cells, but one will appreciate that there is some absoluteamount of these srps above which there is a high risk of cancerrecurrence. This amount could be measured in absolute concentrations andis not necessarily dependent on the relative "internal standard" methodof measurement used to illustrate one embodiment of the invention. Thenumbers obtained using a particular breast cancer cell line to determine"overproduction" will therefore be different from overproduction numbersand cutoff values obtained when other cell comparison standards are usedor if absolute values are determined.

In order to detect and measure srps in human cancers, frozen, storedtumor tissues from 398 primary breast cancers were analyzed for foursrp's by a semi-quantitative Western Blot procedure in a blindedfashion, i.e. without prior knowledge of any tumor characteristics ordisease outcome. Tissues for the quantitation of these proteins wereobtained from the biopsy specimens. Values for estrogen receptor (ER),progesterone receptor (PgR), HER-2/neu oncogene protein (a growth factorreceptor-like transmembrane glycoprotein of 185 kDa) (A. K. Tandon, G.M. Clark, G. C. Chamness, A. Ullrich, W. L. McGuire, J. Clin. Oncol. 7,1120 (1989)), DNA ploidy (a measure of DNA content) (L. G. Dressier, L.C. Seamer, M. A. Owens, G. M. Clark, W. L. McGuire, Cancer 61, 420(1988)), and the 34 kDa mature form of cathepsin D (an estrogen-inducedlysosomal acidic protease) (A. K. Tandon, G. M. Clark, G. C. Chamness,J. M. Chirgwin, W. L. McGuire, N. Engl. J. Med. 322, 297 (1990)) wereavailable on the tumor specimens analyzed.

To account for the variability in content of tumor cells in differentregions within the same specimen, in the first step breast tumors weremechanically pulverized in liquid nitrogen to obtain a uniformdistribution of tumor cells. To further minimize the potential risk ofuneven dilution of tumor cell proteins with stromal proteins, largerquantities (100 mg) than needed (10 mg) were used for proteinextraction. Total proteins were extracted with sodium dodecyl sulfate(A. K. Tandon, G. M. Clark, G. C. Chamness, A. Ullrich, W. L. McGuire,J. Clin. Oncol. 7, 1120 (1989)).

One hundred micrograms of extracted proteins were subjected to WesternBlot analysis using monoclonal antibodies to grp94 (D. P. Edwards, N. L.Weigel, W. T. Schrader, B. W. O'Malley, W. L. McGuire, Biochem. 25, 4427(1984); and D. R. Sargan, M.-J. Tsai, B. W. O'Malley, Biochem. 25, 6252(1986); the monoclonal antibody 9G10 used here reacts with purifiedgrp94 in Western Blot assay, unpublished observation from W. J. Welch'slaboratory), hsp90 (S. Schuh et al., J. Biol. Chem. 260, 14292 (1985)),hsp70 (a gift from W. J. Welch), and hsp27 (D. J. Adams, H. Hajj, D. P.Edwards, R. J. Bjercke, W. L. McGuire, Cancer Res. 43, 4297 (1983)).

Remaining tumor protein extracts were immediately frozen and stored at-70° C. Examples of ten tumors along with an internal reference standardof MCF-7 human breast cancer cells (see below) are shown in FIG. 1. Thelevels of these srp's in individual tumors were calculated in relativeunits per one hundred micrograms tumor protein by the ratio of theintegrated signal in the tumors relative to the MCF-7 internal standard.

The stress response proteins in tumors were measured against the contentof the same stress response proteins in a cell culture of human breastcancer cells. These cells were originally obtained from the MichiganCancer Foundation, and can be purchased from the American Type CultureCollection (ATCC HTB22 MCF-7). In preferred practice the cells arecultured in Eagle's minimum essential medium supplemented withnon-essential amino acids, gentamycin, calf serum, L-glutamine andbovine insulin at near physiological pH, as specified in Example 5.

A second step was to compare the levels of each srp with cancerrecurrence. Levels of all four srp's ranged from undetectable to high.Actual ranges per 100 μg tumor proteins were 0-557 units for grp94;0-173 units for hsp90; 0-862 units for hsp70; and 0-2645 units forhsp27. Distributions of the four srp's under study for node-negative andnode-positive breast cancer patients are shown in FIG. 2. Thedistributions for these srp's in both groups were approximatelylog-normal. Median values of srp's in node-positive versus node-negativebreast tumors were as follows: 37 vs 32 for grp94 (P=0.2); 20 vs 10 forhsp90 (P=0.001); 82 vs 66 for hsp70 (P=0.08); and 48 vs 14 for hsp27(P<0.0001).

Correlations of the srp's with clinical manifestations of breast cancerdisease and its ultimate outcome were determined. Conventionally, themedian value of a given parameter is used to distinguish patients withhigh levels of the parameter from those with low levels. However, medianvalues of all four srp's failed to discriminate these patients into lowand high risks of disease recurrence. Therefore, a biologicallymeaningful cutoff value was sought for different srp's to distinguishpatients at high risk of relapse. A wide range of cutoff values gavestatistically significant separation of disease-free survivalprobabilities in the group of 200 node-negative patients (74 monthsmedian followup for patients still alive) for hsp90 (12 to 59 units),hsp70 (120 to 250 units), and hsp27 (46 to 232 units) (FIG. 2). Forgrp94, unlike hsp's, there was only one single cutpoint at 45 units. Theoptimum cutpoints for hsp90, hsp70, and hsp27 were 32, 217, and 126units, respectively. For 198 node-positive patients the median followupwas 61 months for patients still alive; however, in contrast tonode-negative patients, no cutpoints gave a significant segregation oflow and high risk patients for tumor recurrence (hsp27 marginallyreached statistical significance but the apparent cutpoint range wasextremely limited, FIG. 2).

The interrelationship of four srp's in node-negative breast tumors wasdetermined. Tumors were classified as low or high using the optimumcutpoint for each srp. About fifty percent (101/200) of the tumors werefound to contain low levels of all four srp's, leaving 50% as high foreither one, two, three, or all four srp's. Only 2.5% (5/200) of tumorscontained high levels of all four srp's. Therefore, though these foursrp's were significantly correlated to each other, all were notconcomitantly overproduced in all tumors, perhaps reflecting the diversenature of physiological stress from tumor to tumor. Using the optimumcutpoints, 33.5% of tumors were high for grp94; 24% for hsp90; 15% forhsp70; and 15.5% for hsp27; either alone or in various combinations withthe other three srp's. Data on overlapping incidence of srp's are shownin Table 1, and the correlation values are given in Table 2. A higherincidence of high levels of hsp90 and grp94 indicates that these srp' smay be more sensitive indicators of the biological stress.

                  TABLE 1                                                         ______________________________________                                        Interrelationship of Four Stress Response Proteins                            with Each Other                                                                          Which are also Positive for:                                       % Positive for:                                                                            grp94   hsp90     hsp70 hsp27                                    ______________________________________                                        grp94        100     50        16    17                                       hsp90        67      100       29    35                                       hsp70        34      48        100   55                                       hsp27        35      55        52    100                                      ______________________________________                                         All pairwise comparisons were statistically significant (P < 0.05).           Spearman rank correlation coefficients (r) among these four srp's were al     statistically significant (P < 0.05) and varied from 0.32 to 0.67 (see        Table 2).                                                                

                  TABLE 2                                                         ______________________________________                                        Spearman Rank Correlation Coefficients (r)                                    for the Pairwise Interrelationship of Four Stress Response                    Proteins With Each other                                                              grp 94                                                                              hsp90       hsp70   hsp27                                       ______________________________________                                        grp94                                                                         hsp90     0.67                                                                hsp70     0.44    0.61                                                        hsp27     0.32    0.50        0.63                                            ______________________________________                                         All pairwise comparisons were statistically significant (P < 0.05).      

Association of srp's with other tumor characteristics which arebiological indicators of metastatic potential, hormone responsiveness,or relative histopathologic differentiation was investigated. Data aresummarized in Tables 3A and 3B. All four srp's were directly associatedwith high levels of cathepsin D, an estrogen-induced lysosomal proteasesupposed to be a marker of metastatic potential (hsp27 failed to reachstatistical significance). Similar results were found using cathepsin Ddirectly as a clinical discriminator in these sets of node-negative andnode-positive patients (A. K. Tandon, G. M. Clark, G. C. Chamness, J. M.Chirgwin, W. L. McGuire, N. Engl. J. Med. 322, 297 (1990)).

In addition to cathepsin D, the following correlations/associations wereobserved: hsp27 and hsp70 with estrogen and progesterone receptorstatus; hsp90 and grp94 with aneuploidy; hsp70, hsp90, and grp94 withnuclear grade; and hsp27 and hsp90 with HER-2/neu oncogene protein. Nosignificant correlations were found with tumor size or patient age.

The data in Tables 3A and 3B were obtained by analysis of tumors from398 breast cancer patients ranging in age from 26 to 82 years with amedian of 58 years. Tumor grp94≧45 units; hsp90≧32 units; hsp70≧217units; and hsp27≧126 units per 100 μg of total tumor proteins werecategorized as high (positive). Nodal status was considered negativewhen no lymph nodes contained tumor cells and positive if one or morenodes showed the presence of malignant cells. Tumor size (largestdiameter) was recorded at the time of surgery. Levels of estrogenreceptor (ER) and progesterone receptor (PgR) in fresh tumor cytosolswere determined by standard methods (W. L. McGuire, M. De La Garza, G.C. Chamness, Cancer Res. 37, 637 (1977); B. Powell, R. E. Garola, G. C.Chamness, W. L. McGuire, ibid, 39, 1678 (1979)) and are expressed asfmoles per mg cytosolic proteins. Ploidy (DNA content) was determined byflow cytometric analysis (L. G. Dressier, L. C. Seamer, M. A. Owens, G.M. Clark, W. L. McGuire, Cancer 61, 420 (1988)). Levels of the HER-2/neuoncogene protein and the 34 kDa mature form of cathepsin D werequantitated by Western blotting and densitometry methods (A. K. Tandon,G. M. Clark, G. C. Chamness, A. Ullrich, W. L. McGuire, J. Clin. Oncol.7, 1120 (1989); A. K. Tandon, G. M. Clark, G. C. Chamness, J. M.Chirgwin, W. L. McGuire, N. Engl. J. Med. 322, 297 (1990)). Analysis ofdata for association between srp's and other characteristics in breastcancer was performed using two-way contingency tables and non-parametriccorrelation coefficients.

                                      TABLE 3A                                    __________________________________________________________________________    Relationship Between Stress Response Proteins and Other Clinical              Characteristics                                                               in Node-Negative Breast cancer                                                          grp94    hsp90    hsp70    hsp27                                    Characteristic n                                                                        % grp+                                                                             P   % hsp+                                                                             P   % hsp+                                                                             P   % hsp+                                                                             P                                   __________________________________________________________________________    Cathepsin D                                                                   < 75 units                                                                              136  24  17       9        13                                                      0.0006   0.0006   0.009    0.09                                ≧5 units                                                                         64   48  39       27       22                                       Nuclear Grade                                                                 1  6      33   0        0        0                                            2 103     28   0.06                                                                              18   0.002                                                                             12   0.08                                                                              14   0.21                                3  70     46   40       23       21                                           Histologic Grade                                                              1  6      33   0        0        0                                            2 111     30   0.12                                                                              24   0.17                                                                              14   0.39                                                                              17   0.54                                3  62     45   32       19       16                                           Estrogen Receptor                                                             ≧3 fmol/mg                                                                       142  30  26       18       18                                                      0.25     0.29     0.02     0.09                                <3 fmol/mg                                                                              58   38  19       5        9                                        Progesterone                                                                  Receptor                                                                      ≧5 fmol/mg                                                                       99   27  23       24       20                                                      0.16     0.80     0.0001   0.07                                <5 fmol/mg                                                                              101  37  25       5        11                                       Tumor Size                                                                    ≦2 cm                                                                            73   30  21       18       12                                                      0.67     0.39     0.31     0.35                                >2 cm     127  33  26       13       17                                       Ploidy                                                                        Diploid   72   24  14       10       13                                                      0.03     0.008    0.12     0.31                                Aneuploid 116  39  31       18       18                                       Age                                                                           ≧50 years                                                                        137  33  27       18       15                                                      0.71     0.14     0.07     0.92                                <50 years 63   30  17       8        16                                       HER-2/neu                                                                     <100 units                                                                              171  32  21       16       13                                                      0.66     0.01     0.23     0.04                                ≧100 units                                                                       28   36  43       7        29                                       __________________________________________________________________________

                                      TABLE 3B                                    __________________________________________________________________________    Relationship Between Stress Response Proteins and Other Clinical              Characteristics                                                               in Node-Positive Breast Cancer                                                          grp94    hsp90    hsp70    hsp27                                    Characteristic n                                                                        % grp+                                                                             P   % hsp+                                                                             P   % hsp+                                                                             P   % hsp+                                                                             P                                   __________________________________________________________________________    Cathepsin D                                                                   <75 units 101  64  30       15       22                                                      0.001    0.19     0.51     0.08                                ≧75 units                                                                        98   85  39       18       33                                       Estrogen Receptor                                                             ≧3 fmol/mg                                                                       142  75  35       21       35                                                      0.89     0.94     0.007    0.0002                              <3 fmol/mg                                                                              57   74  34       5        9                                        Progesterone                                                                  Receptor                                                                      ≧5 fmol/mg                                                                       100  76  34       24       36                                                      0.60     0.92     0.005    0.005                               <5 fmol/mg                                                                              99   73  35       9        18                                       Tumor Size                                                                    <2 cm     34   76  26       12       32                                                      0.76     0.29     0.41     0.45                                ≧2 cm                                                                            165  74  36       18       26                                       Age                                                                           ≧50 years                                                                        129  74  37       21       30                                                      0.98     0.34     0.03     0.18                                <50 years 70   74  30       9        21                                       HER-2/neu                                                                     <100 units                                                                              163  36  32       18       30                                                      0.21     0.10     0.07     0.07                                ≧100 units                                                                       34   50  47       6        15                                       No. of Positive                                                               Nodes                                                                         1-3  74   39   38       14       28                                                          0.82     0.42     0.37     0.76                                >3 125    38   32       18       26                                           __________________________________________________________________________

The association of each srp with the likelihood of development ofrecurrent breast cancer was studied. Kaplan-Meier analyses of time torecurrence for the node-negative patients are shown in FIG. 3. Patientswith higher levels of grp94, hsp90, hsp70, or hsp27 in primary tumorswere at a greater risk of developing early recurrent cancer compared tothose with lower levels of these proteins.

Both hsp27 and hsp70 distinguished patients with different recurrencerates within 18 months of surgical removal of tumor. Hsp90 separated thetwo groups of patients within a few months of primary treatment.

Kaplan-Meier recurrence curves were constructed based upon the higherlevels of any one of the four, two of the four, three of the four, orall four srp's in the tumor, and compared with those containing only lowlevels of all four srp's (FIG. 4). Concomitant presence of more than onehigh srp in the tumor was highly indicative of early recurrence ofdisease. The five-year actuarial recurrence was 24% to 27% in patientswith low levels of all four srp's or with high level of only one of thefour srp's, 47% in patients with high levels of two of four srp's, and8% to 80% in patients with high levels of three or all four srp's. Dataare shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Five-Year Actuarial Recurrence of Node-Negative                               Breast Cancer Patients as a Function of                                       Stress Response Proteins (srp'a)                                              No. of Positive                                                                              No. of   % Recurrence                                          srp's          Patients ± S.E.                                             ______________________________________                                        None           101      24 ± 4                                             One of four    48       27 ± 7                                             Two of four    34       47 ± 9                                             Three of four  12       78 ± 13                                            All four        5       80 ± 18                                            ______________________________________                                         High levels of srp's, defined in Table 3A, are denoted as positive.      

Stress response proteins and several other tumor characteristics weresubjected to a multivariate analysis to explore their independentcontribution in predicting disease-free survival probability innode-negative breast cancer patients. Ploidy, nuclear grade, andhistologic grade were not included in this analysis since values forthese variables were not available for many of the tumors.

A partially non-parametric regression model was used to evaluate thepredictive power of various combinations and interactions of prognosticfactors in a multivariate manner (N. E. Breslow, Int. Stat. Rev. 43, 45(1975); D. R. Cox, J.R. Stat. Soc. B. 34, 187 (1972); J. D. Kalbfleischand R. L. Prentice, The Statistical Analysis of Failure Time Data, NewYork, Wiley, 1980)). Variables were entered stepwise and the relativerisks are presented only for the retained variables. Median clinicalfollowup time for patients still living was 74 months with a range of 29to 154 months.

Stress response (P=0.001) and cathepsin D (P=0.004) appeared asindependent predictors of early recurrence, as indicated in Table 5.Relative risk for stress response was 1.4 per additional high srp.Inclusion of ploidy in multivariate analysis did not affect thedominance of srp's and cathepsin D. However, when patients with zero orone high srp were grouped and compared with all patients with two ormore high srp's, the statistical significance of srp's was slightlyweakened.

                  TABLE 5                                                         ______________________________________                                        Stress Response Proteins and Other Tumor Characteristics in                   Multivariate Disease-Free Survival in 199 Node-Negative                       Breast Cancer Patients                                                        Relative                                                                      Factor              Multivariate                                              Risk                P-Value                                                   ______________________________________                                        Stress Response Proteins                                                                           0.001  1.4*                                              Cathepsin D          0.004  2.1                                               Patient Age         0.08                                                      Progesterone Receptor                                                                             0.11                                                      HER-2/neu Protein   0.13                                                      Estrogen Receptor   0.61                                                      Tumor Size          0.91                                                      ______________________________________                                         *srp's were considered as an ordinal variable (0 to 4+) so the relative       risk is shown as per positive (i.e. high level) srp.                     

The four srp's analyzed are frequently present in primary breast tumorsand are found to be associated with clinical variables suggesting lymphnode invasion, tumor aggressiveness, hormone-responsiveness, andhistopathologic de-differentiation. Breast cancer patients whose tumorscontained lower levels of srp's had a significantly greater likelihoodof surviving free of recurrence (second tumor) than patients with higherlevels of srp's. Simultaneous occurrence of high levels of more than onesrp is a stronger indicator of early disease-recurrence. In multivariateanalysis, stress response joins cathepsin D in predicting early tumormetastasis.

Kits useful in the present invention comprise a carrier havingcompartments to receive several closed containers, the number dependingon the specific reagents required for the method of analysis. All kitswould provide a first container means comprising a stress responseprotein standard which contains known levels of hsp27, 70, 90 and grp94; a second container means comprising a negative control breast tumorextract, a third container means comprising a positive control breasttumor extract, and a fourth container means comprising four monoclonalantibodies to the four stress response proteins. Methods of stressresponse protein determination could be based on Western Blot, ELISA, orimmunohistochemical analysis.

In a Western Blot procedure, a breast tumor sample is pulverized inliquid nitrogen to obtain a uniform distribution of cells, extracted,and total protein determined. After polyacrylamide gel electrophoresisand incubation with the appropriate antibodies, the sample srp bands aremeasured by densitometry and reported as a ratio against the srp bandsof the included standards. The ratios are related to "cutoff" values asdescribed earlier which indicate tumor recurrence risk.

An immunohistochemical method would be useful in kit form. The tumorsample is sectioned and fixed on an adhesive-coated slide which can beprovided with the kit. Six sections are incubated with a normal animalserum, then four of these are treated with four different monoclonalantibodies to the stress response proteins and two with antibodynegative controls. The antibodies are also applied to four controlslides provided with the kit. All sections are incubated withbiotinylated second antibody, incubated with avidin and biotinylatedperoxidase, then incubated with diaminobenzidine and osmium tetroxide,and stained with methyl green. Positive staining shows brown colorationin the cytoplasm and is quantitated by determining both the fraction ofstained tumor cells in several fields and the degree of their positivityto develop an H-score, as is presently done for a number of otherproteins.

An ELISA assay kit would provide the monoclonal antibodies to the stressresponse proteins, the stress response protein standards, positive andnegative breast tumor cytosol controls, and a second set of monoclonalantibodies to the stress response proteins coupled to horseradishperoxidase.

EXAMPLE 1

Detection and Measurement of Stress Response Proteins

Total proteins from tumor tissues or cell pellets were extracted withsodium dodecyl sulfate (SDS). Protein concentration in the SDS-extractwas determined by the BCA method (P. K. Smith et al., Anal. Biochem.150, 76 (1985)). SDS-extracted proteins were mixed with a sample bufferto achieve a final concentration of 135 mM Tris (pH 6.8), 2% SDS, 10%glycerol, 5% dithiothreitol, and 0.01% pyronin dye and the samples wereheated in a boiling water bath for five minutes.

Tumor proteins (100 μg) were resolved on 10% polyacrylamide verticalslab gels using 3% stacking gel. An SDS extract of MCF-human breastcancer cells was included at three concentrations (100 μg, 50 μg, and 25μg protein, corresponding to 100, 50 and 25 arbitrary units of eachstress response protein) in each gel as an internal reference standard.

Transfer of separated proteins onto 0.2μ nitroscreens (DuPont) wasperformed at 200 mAmp for 16 hours at 4° C. Following blocking ofnon-specific sites with 5% condensed milk powder (Carnation) for 1 hour,blots were incubated with culture supernatant of a hybridoma clonesecreting rat monoclonal antibody (MAb) to grp94 (clone 9G10) overnightat 4° C. ¹²⁵ I-labeled sheep anti-rat IgG (100,000 cpm/ml; AmershamCorp) was used as the second antibody. After washing, the blots wereexposed for 20-24 hours to X-OMAT X-ray film (Kodak) at -70° C. The samemembranes were next reacted with purified mouse MAb to hsp70 (C92) at 1μg/ml followed by ¹²⁵ I-labeled sheep anti-mouse IgG (100,000 cpm/ml;Amersham Corp) and exposed to X-ray film as described above, and thenincubated with a mouse MAb to hsp27 (1 μg/ml) again followed byradiolabeled second antibody and x-ray film autoradiography.

For measuring hsp90, another set of gels was run under the sameconditions as described above and membranes were incubated with a mouseMAb (AC88, 1 μg/ml) followed by incubation with ¹²⁵ I-labeled anti-mouseIgG second antibody and exposure to X-ray film. The level of srp's inindividual tumors was quantitated by densitometric scanning of thepertinent band on the autoradiogram in a Beckman DU-7 spectrophotometer,and expressed in relative units by comparison with the MCF-7 internalreference standard.

The srp values in FIG. 1 for ten tumors (1 to 10) were: 33, 85, 19, 46,70, 74, 54, 54, 27, 108 for grp94; 23, 76, 13, 50, 32, 58, 6, 16, 4, 12for hsp90; 63, 261, 75, 119, 46, 211, 46, 211, 86, 86 for hsp70; and194, 245, 41, 179, 91, 112, 5, 61, 139, 31 for hsp27.

EXAMPLE 2

Determination of Optimum Cutoff Values

Cutoff values for the four srp's that best distinguish patients at highrisk for relapse were established by determining P-values fordisease-free survival using each possible cutoff value. FIG. 2graphically displays these P-values for both. node-positive andnode-negative breast cancer patients. A horizontal line is drawn at theP=0.05 level to show statistical significance. Upward arrows indicatethe optimum cutpoints. Range and optimum cutoff values for the foursrp's in node-negative breast cancer patients are given below:

    ______________________________________                                        Range and Optimum Cutoff Values for srp's                                     in Breast Tumors                                                              Cutpoint Stress Response                                                                             Cutoff Range                                                                             Optimum                                     ______________________________________                                        1.       grp94         45 units    45 units                                   2.       hsp90         12-59 units                                                                               32 units                                   3.       hsp70         120-250 units                                                                            217 units                                   4.       hsp27          46-232 units                                                                            126 units                                   ______________________________________                                    

EXAMPLE 3 Determination of Recurrence Relative to Stress ResponseProtein Level

Based on optimum cutoff determinations, high srp's are defined asfollows: grp94≧45 units, hsp90≧32 units, hsp70≧217 units, and hsp27≧126units per 100 μg tumor proteins. Patients were followed for disease-freesurvival. Any post-surgical appearance of malignancy either near to ordistant from the operated breast was considered as recurrence ofdisease. Survival curves were constructed and shown in FIG. 3 (E. L.Kaplan and P. Meier, J. Am. Stat. Assoc. 53, 457 (1958)) and the logrank test for censored survival data was used to test the statisticalsignificance of difference between the curves (N. Mantel, CancerChemother. Rep. 50, 163 (1966)). All computations were done with theBiomedical Computer Programs-P series. The recurrence curves were basedon a clinical followup period of 29 to 154 months, with a median of 74months for patients still alive at the time of analysis. Values belowthe X-axis indicate the number of patients at risk at the intervalshown.

EXAMPLE 4 Determination of Disease-Free survival Relative to Number ofHigh Level Stress Response Proteins

Survival curves as a function of the number of srp's found to exceedtheir cutoff values are shown in FIG. 4. Positive (high) levels are thesame as described in Example 3. Median followup was 74 months. Valuesbelow the X-axis show the number of patients at risk at the indicatedtime interval. Statistical significance (P-value) for pairwisecomparisons between groups of patients separated according to the numberof their high level srp's is given below.

    ______________________________________                                                      One    Two                                                             No srp+                                                                              srp+   srp+   Three srp+                                                                             Four srp+                                ______________________________________                                        No srp+                                                                       One srp+ 0.6                                                                  Two srp+ 0.01     0.1                                                         Three srp+                                                                             0.0006   0.009  0.2                                                  Four srp+                                                                              0.0001   0.002  0.04 0.2                                             ______________________________________                                    

EXAMPLE 5 Standard for the Measurement of Stress Response Proteins

MCF-7 human breast cancer cells (originally obtained from the MichiganCancer Foundation), ATCC HTB22 MCF-7 were cultured in Eagle's minimumessential medium (MEM) supplemented with 10 mM HEPES, 1% non-essentialamino acids (Gibco), 2 mM L-glutamine (Gibco), 25 μg/ml gentamycin(Irvine Scientific), 6 ng/ml bovine insulin, and 5% calf serum (K.C.Biologicals). Sodium bicarbonate (0.2%) was added to adjust the final pHto approximately 7.2. Cells were allowed to grow at 37° C. in anatmosphere containing 5% CO₂. Logarithmically growing MCF-7 cells closeto confluency (75-100%) were harvested by a brief incubation with 1 mMEDTA in phosphate buffered saline (PBS). Cells were washed twice withPBS, and pelleted. Cell pellets were exposed to 5% sodium dodecylsulfate (SDS), vortexed, and kept in a boiling water bath for 5 minutes,revortexed, and allowed to cool to room temperature for about 15minutes. Clear supernatant was collected after spinning the tubes in acentrifuge. Protein concentration in the SDS extracts was determined bythe BCA method (P. K. Smith et al., Anal, Biochem. 150, 76 (1985)). Forthe estimation of quantity of stress response proteins (srp's) in breasttumors by Western Blot, this SDS extract of MCF-7 human breast cancercells was included in each gel at three concentrations (100 μg, 50 μg,and 25 μg protein, corresponding to 100, 50, and 25 units). The level ofsrp's in breast tumors was expressed in units relative to this standard.

The following examples, 6, 7, and 8, illustrate how stress responseproteins could be analyzed by convenient kit means. The examples havenot been tested using precisely the steps outlined but are illustrativeof how such kits would be used.

EXAMPLE 6 This Example Illustrates the Steps that Could be Used in aWestern Blot Kit for Stress Response Protein Determination

Mechanically pulverize the breast tumor specimen in liquid nitrogen toobtain a uniform distribution of tumor cells. Add 1 ml of 5% sodiumdodecyl sulfate to 100 mg of tumor powder and vortex. Place the tube ina boiling water bath for 5 min and vortex. Centrifuge the tube at13,000×g for 2 min and determine protein concentration in the clearsupernatant using BCA reagents by mixing reagent A and B in a 50:1ratio. Add 1 ml of this mixture to a 100 μl aliquot of test dilutedsample or protein standard.

Incubate at room temperature for 1 hour, read absorbance at 562 nm, andcalculate protein concentration by interpolation on the protein standardcurve constructed based on the protein standard used in each experiment.Electrophorese 100 μg of solubilized tumor proteins on 10%polyacrylamide Laemmli gel under denaturing, reducing conditions. Oneach gel load the stress response proteins standard (vial 1) and tumorextracts from vial 2A and 2B as positive and negative controls.Electrically transfer proteins from the gel to nitroscreen filter(Towbin's procedure) using 200 mAmp current for 16 hours in the cold.Block nitroscreen by incubation for 1 hour at room temperature with 5%evaporated milk in PBS. Incubate nitroscreen with 1:50 fold dilution(prepared in 5% milk) of antibodies to stress-response proteins (vial 3)for 2 hours at room temperature with gentle shaking. Wash nitroscreen 3times for 5 minutes each with phosphate buffered saline (PBS) on ashaker at room temperature. Incubate nitroscreen with 1:500 folddilution (in 5% milk) of radioactive second antibody (vial 4) for 1 hourat room temperature with gentle shaking. Wash nitroscreen 3 times for 5minutes each with PBS on a shaker at room temperature. Exposenitroscreen overnight to x-ray film at -70° C. Develop film and estimatethe amount of four srp's in tumor specimen by densitometry of therelevant bands and calculation of ratios with the srp standard bands.High levels of srp's are defined as follows: grp94≧45 units, hsp90≧32units; hsp70≧217 units, and hsp27≧126 units per 100 μg of tumorproteins.

EXAMPLE 7 This Example Illustrates the Steps that Could be Used in theImmunohistochemical Determination of Stress Response Proteins by a KitMethod

Cut six 5-micron sections from the frozen OCT block of a breast tumorspecimen and place each section on a separate adhesive-coated microscopeslide provided with the kit. Air dry tissue sections for 30 minutes atroom temperature (RT). Dip slides in -20° C. acetone for 5 minutes. (Forformalin-fixed paraffin-embedded tumors, cut six 5-micron sections fromthe paraffin block. Bake at 60° C. for 30 minutes in an oven. Dip slidesin xylene 2-times for 5 minutes each. Place slides 2-times in 100%alcohol for 5 minutes each. The rest of the procedure is common to bothfrozen and fixed tumors. Wash 2-times with PBS for 2 minutes each. Placeslides for 30 minutes in PBS containing 0.1% H₂ O₂ and 0.1% sodiumazide. Wash 2-times with PBS for 2 minutes each. Cover tissue sectionswith 10% normal goat serum (vial 1) for 30 minutes at RT. Drain thesolution and incubate four sections of the test tumor with fourdifferent monoclonal antibodies to srp's (vial 2A, 2B, 2C, 2D) at thedilution indicated on each vial.

Similarly apply these antibodies to four control slides provided withthe kit (control slides of breast tumor sections showing positivestaining for srp's). Treat two remaining sections of the test tumor withthe antibody negative controls (3A, 3B). Incubate all ten slides in acovered humidity chamber for 3 hours at RT. Wash 2-times with PBS for 2minutes each. Incubate sections for 30 minutes at RT with appropriatebiotinylated second antibody (4A or 4B) at the indicated dilution(sections treated with grp94 antibody or normal rat antibody control aretreated with anti-rat second antibody (4A), while all other sections aretreated with anti-mouse second antibody (4B)). Wash 2-times with PBS for2 minutes each.

Mix reagents from vial 5A and 5B as indicated on the vials to prepareavidin-biotin-peroxidase complex and apply to the sections for 30minutes at RT. Wash 2-times with PBS for 2 minutes each. Incubatesections with diaminobenzidine (vial 6) dissolved as instructed in PBScontaining 0.03% H₂ O₂. Wash 2-times with PBS for 2 minutes each. Placeslides in osmium tetroxide solution (vial 7) for 30 seconds. Wash withdeionized water for 2 minutes and place in 0.5% methyl green for 2minutes. Wash with deionized water for 2 minutes and dehydrate tissue bydipping in increasing concentrations of alcohol and finally in xylene.Cover with permount, place cover slip on the tissue and dry. View tissuesections under a microscope. Positive staining shows brown coloration inthe cytoplasm.

EXAMPLE 8 This Example is Illustrative of Steps that Would be Utilizedin an ELISA Kit Determination of Stress Response Proteins

Coat 96-well microtiter places with a monoclonal antibody to srp(monoclonal antibodies to four different srp's are provided with thekit; vial 1A-1D). Incubate plates overnight at 4° C. with the antibodysolution (100 μl/well) at 5 μg/ml in sodium carbonate buffer pH 9.6, oralternatively antibody-coated microtiter plates can be provided with akit. Wash plates with PBS 3-times and incubate with 1% BSA in PBS for 1hour at room temperature (RT). Wash with PBS 6-times. Dispense 100μl/well test breast tumor cytosols, positive and negative controls (vial2A, 2B) and standards (vial 3A-3D). Incubate at RT for 2 hours withgentle agitation. Wash with PBS 6-times. Incubate with 100 μl/well of asecond set of anti-srp monoclonal antibodies labelled with horseradishperoxidase (vial 4A-4D) for 1 hour at RT. Wash with PBS 6-times. Add 100μl/well of ortho-phenylene diamine (OPD) solution. Incubate in the darkat RT for 15 minutes. Stop reaction by adding 100 μl/well sulfuric acidand record absorbance at 490 nm.

Construct standard curve by plotting absorbance against srpconcentration. Calculate srp concentration in test cytosols byinterpolation of their absorbance on the standard curve.

EXAMPLE 9 This Example Illustrates the Procedure Contemplated by theApplicants as useful in Determining the Level of Stress Response Proteinby Measuring mRNA Levels

The example is illustrated with hsp90, but would be appropriate forother hsps, including hsp27, hsp70 and grp94. Amino acid sequences forthese hsps are known, thus DNA sequences are readily determined and cDNAobtained by well-known cloning procedures. Details of the general stepsmay vary but generally procedures for determining mRNA levels areroutinely used by those of skill in the art.

Determination of HSP90 Levels in Tumor Tissue

Pulverized tumor tissue samples are homogenized in guanidineisothiocyanate and total cellular RNA extracted by standard extractiontechniques for RNA. After precipitation with ethanol, RNA pellets areresuspended in water and checked for absorbance at 320 nm. The procedureis repeated as necessary to obtain preparations free of proteincontamination, as indicated by lack of absorbance at 320 nm. RNAconcentrations are adjusted to 1 μg/ml. Northern blot hybridization isperformed using labeled riboprobes. The probes for hsp90 are prepared asdescribed (Hickey, L., Brandon, L. E., Smale, G., LLoyd, D. and Weber,L. A., Mol. Cell 9, 2615-2625 (1989) from single-stranded probesobtained from fragments cloned in M13 using standard hybridizationprocedures. Probes are labelled with [³⁵ S]UTP. Hybridization isquantitated by densitometry.

The references cited within the text are incorporated herein byreference to the extent that they supplement, explain, provide abackground for or teach methodology, techniques and/or compositionsemployed herein.

We claim:
 1. An in vitro method for prognosis of disease-free survivalof individuals having a breast cancer tumor, comprising determining ifat least two selected stress response proteins are overproduced in asample of such a tumor, such an overproduction correlating negativelywith disease-free survival.
 2. The method of claim 1 whereinoverproduction is determined by measuring the levels of mRNAs whichencode the selected stress response proteins.
 3. The method of claim 1wherein overproduction is determined by measuring protein levels forselected stress response proteins.
 4. The method of claim 1 whereinoverproduction is determined by determining the number of stressresponse proteins that are detectable in breast tumor tissue or inblood, serum or plasma samples.
 5. The method of claim 1, furtherdefined asdetermining the level of production of said selected stressresponse proteins; and determining if said level of production comprisesan overproduction that is above a basal level.
 6. The method of claim 5wherein basal levels of the selected stress response proteins aredetermined relative to levels of said proteins produced by culturedcells that are capable of producing stress response proteins in vitro.7. The method of claim 6 wherein the cultured cells are cancer cells,normal cells, genetically engineered cells or progeny of said cells. 8.An in vitro method for predicting the risk of tumor recurrence inindividuals having a selected tumor, comprising determining if at leasttwo selected stress response proteins are overproduced in a sample ofsuch a tumor, such an overproduction correlating positively with alikelihood of tumor recurrence.
 9. The method of claim 8, furtherdefined as comprising:determining an overproduction level for selectedstress response proteins, said level being in excess of a minimum amountstatistically determined to related tumor recurrence; determining thelevels of the selected stress response proteins in tumor sample; andpredicting a risk of tumor recurrence wherein an overproduction level ofat least two stress response proteins in the tumor sample is positivelyassociated with the likelihood of tumor recurrence.
 10. The methodrecited in claim 1, 5 or 8 wherein the stress response proteins havemolecular weight of 94 kDa, 80 to 90 kDa, 68 to 74 kDa, or 18 to 30 kDa.11. The method of claim 1, 5, or 8 wherein at least two of the followingstress response proteins are assessed: hsp27, hsp70, hsp90 and grp94.12. The method of claim 1 wherein the breast tumor is in a node negativebreast cancer patient.
 13. The method of claim 11 wherein determiningthe stress response proteins comprises comparing levels of the stressresponse proteins in said samples with levels of said stress responseproteins from breast cell cancer line MCF-7, wherein a negativecorrelation with disease free probability exists when at least two ofthe following are determined:sample grp94 is at least 45% of grp94 incancer cell line standard MCF-7 from an amount of protein in saidstandard equivalent to that of the tissue sample; sample hsp27 is atleast 46% of hsp27 in cancer cell line standard MCF-7 from an amount ofprotein in said standard equivalent to that of the tissue sample; samplehsp90 is at least 12% of hsp90 in cancer cell line standard MCF-7 froman amount of protein in said standard equivalent to that of the tissuesample; or sample hsp70 is at least 120% of hsp70 in cancer cell linestandard MCF-7 from an amount of protein in said standard equivalent tothat of the tissue sample.
 14. The method of claim 11 wherein an optimalcutoff overproduction level obtained relative to standard breast cellline MCF-7 comprises:(a) 126 units per 100 μg tumor proteins for hsp27;(b) 217 units per 100 μg tumor proteins for hsp70; (c) 32 units per 100μg tumor proteins for hsp90; or (d) 45 units per 100 μg tumor proteinsfor grp94.
 15. A kit for the immunologic assessment of breast tumorrecurrence or survival, the kit comprising:(a) at least one aliquotedantibody which specifically binds a stress response protein selectedfrom the group consisting of hsp90, hsp27, and grp94 provided thatantibodies which specificially bind hsp27 are included with at least oneother of said antibodies; and (b) an immunologic detection reagent. 16.The kit of claim 15, wherein the immunologic detection reagent comprisesa radioisotopic, fluorometric, enzymatic or colorimetric label.
 17. Thekit of claim 16, wherein said label is associated with a secondaryantibody.
 18. The kit of claim 15, specifically adapted for Western Blotdetection and measurement of tumor-associated stress response proteins,the kit comprising:a carrier being compartmentalized to receive one ormore container means in close confinement therein; a first containermeans comprising a stress response protein standard; a second containermeans comprising a negative control; a third container means comprisinga positive control; a fourth container means comprising one or moreseparate compartments each comprising a first antibody whichspecifically binds a stress response protein selected from the groupconsisting of hsp70, hsp90, hsp27 grp94 provided that antibodies whichspecifically bind hsp27 are included with at least one other of saidantibodies; and a fifth container means comprising one or more separatecompartments each comprising labeled second antibodies whichspecifically bind the first antibodies.
 19. The kit of claim 18 whereinthe stress response protein standard, the positive and negativecontrols, and the antibodies are in lyophilized or liquid form.
 20. Thekit of claim 15, specifically adapted for use in the immunohistochemicaldetermination of stress response proteins in tumor tissue comprising:acarrier being compartmentalized to receive one or more container meansin close confinement therein; a first container means comprising anormal serum from a first animal; a second container means comprisingseparate vials each comprising antibodies derived from a second or thirdanimal, each antibody being directed to a different stress responseproteins; a third container means comprising two separate vials, thefirst vial comprising a biotinylated antibody from a fourth animalspecifically binding antibodies from the second animal, the second vialcomprising a biotinylated antibody from a first, fourth, or fifth animalspecifically binding antibodies from the third animal; a fourthcontainer means comprising an antibody negative control obtained fromthe second animal; a fifth container means comprising an antibodynegative control obtained from the third animal; and a sixth containermeans comprising control slides with tumor sections.
 21. The kit ofclaim 20 wherein the biotinylated antibody from a fourth animal can alsobe from the first animal.
 22. The kit of claim 21 wherein thebiotinylated second antibodies comprising the third container areanti-rat and anti-mouse biotinylated antibodies.
 23. The kit of claim 20wherein the antibody negative controls comprising the fourth and fifthcontainer are normal rat and normal mouse IgG.
 24. An in vitro methodfor prognosis of disease free survival in breast cancer patients havingor once having a tumor, comprising determining if at least two selectedstress response proteins or related metabolites are overproduced in asample of such tumor or in a blood, serum., or plasma sample, such anoverproduction correlating negatively with disease-free survival.